The invention relates to methods for immobilization of glycocompounds and glycoconjugates, and in particular immunoglobulins, onto solid supports for use in affinity chromatography.
Affinity chromatography is a well-established technique used to purify a variety of materials of biological and chemical interest, for example proteins. The system relies on the unique interaction between an immobilized affinity ligand and the molecule of interest. Since the introduction of affinity chromatography methods, immobilized ligands on solid supports have found application in the purification of receptors, enzymes and antibodies. Immobilized antibodies and binding proteins have also been used as biosensors and in bioreactors. In view of the particular importance of immunoglobulins in research, diagnostic procedures and bioseparations, much of the work in this field has focused on methods for binding antibodies; while the following discussion relates in particular to immunoglobulins, it should be understood that many of the principles involved are of more general applicability to a wide class of potential ligands.
Most of the published reports relating to the immobilization of ligands, such as in particular antibodies, have emphasized the central importance of the stability and retention of specific binding characteristics of the immobilized species. To this end, a great deal of research into the chemistry of binding has taken place and a variety of activated supports are now commercially available. Antibodies have been bound to a variety of solid supports, including matrices prepared for covalent binding by CNBr activation [Cuatrecasas, P., "Protein Purification by Affinity Chromatography," J. Biol. Chem. 245, 3059-3065 (1970)] or periodate activation [Ferrua, B., et al., "Coupling of Gamma-globulin to Microcrystalline Cellulose by Periodate Oxidation," J. Immunol. Methods. 25, 49-53 (1979)], and matrices containing N-hydroxysuccinimide esters [Laporte, D. C., et al., "Inhibition of Escherichia Coli Growth and Respiration by Polymyxin B Covalently Attached to Agarose Beads," Biochemistry 16, 1642-1648 (1977); Wilchek, M. et al., "Limitations of N-Hydroxysuccinimide Esters in Affinity Chromatography and Protein Immobilization," Biochemistry 26, 2155-2161 (1987)].
Generally, the established procedures covalently couple antibodies to solid supports through the reactive amino group of an amino acid residue. Previously, the use of various solid phase supports for antibody immobilization has been reported [Little, M. C., et al., "Enhanced Antigen Binding to IgG Molecules Immobilized to a Chromatographic Support via Their Fc Domains," BioChromatography 3, 156-159 (1988); Matson, R. S., and M. C. Little, "Strategy for the Immobilization of Monoclonal Antibodies on Solid Phase Supports," J. Chromatogr. 458, 67-77 (1988)].
However, this method of binding biological materials via amino acid residues has several inherent problems. For instance, if lysine groups are present on the surface of the antibody molecule, multi-site attachment frequently occurs and the antibody may lose activity. In addition, multiple orientations of the antibody on the gel surface are possible, depending on how many and which lysine groups bind to the gel.
Moreover, because of the lack of specificity in the binding reaction when coupling occurs through the reactive amino groups of amino acid residues, the antibody may be bound at a site within, or in close proximity to, a ligand binding site. This further reduces the specific binding activity of the ligand.
Many proteins that could be bound to solid supports for use in affinity techniques are glycoproteins. The oligosaccharide moieties of these glycoproteins are usually located at sites away from the ligand binding site and are not believed to be involved in the binding of bioactive molecules [O'Shannessy D. J., and R. H. Quarles, "Labeling of the Oligosaccharide Moieties of Immunoglobulins," J. Immunol. Methods 99, 153-161 (1987); Williams, D. G., "Comparison of Three Conjugation Procedures for the Formation of Tracers for Use in Enzyme Immunoassays," J. Immunol. Methods 72, 261-268 (1984); Tsang, V. C. M., et al., "Quantitative Capacities of Glutaraldehyde and Sodium m-Periodate Coupled Peroxidase-Anti-Human IgG Conjugates in Enzyme-Linked Immunoassays," J. Immunol. Methods. 70, 91-100 (1984)]. Therefore, the oligosaccharide moieties of these glycoproteins may be specifically modified with little or no effect on the ligand binding properties of the glycoproteins.
A particularly advantageous technique for immobilization of glycocompounds and glycoconjugates, and in particular glycoproteins such as immunoglobulins, involves the modification of the oligosaccharide moieties so as to introduce therein suitable reactive groups for formation of covalent bonds with complementary reactive groups on a matrix material. Thus, for example, hydrazide groups (which react with aldehydes resulting from the oxidation of the oligosaccharide moieties of glycoconjugates) have been successfully exploited for the preparation of immunoglobulins bound to a solid support [O'Shannessy, D. J. and Hoffman, W. L., "Site-Directed Immobilization of Glycoproteins on Hydrazide-Containing Solid Supports," Biotechnol. & Appl. Biochem. 9, 488-496 (1987); Hoffman, W. L. and O'Shannessy, D. J., "Site-specific Immobilization of Antibodies by their Oligosaccharide Moieties to New Hydrazide Derivatized Solid Supports," J. Immunological Methods 112, 113-120 (1988); Turkova, J. et al., "Carbohydrates as a Tool for Oriented Immobilization of Antigens and Antibodies," J. Chromatography 500, 585-593 (1990)].
More recently, the performance of hydrazide AvidGel.TM. Ax (BioProbe International) as a support matrix for immobilization of IgG for use in affinity chromatography has been examined [Cress, M. and T. Ngo, "Site Specific Immobilization of Immunoglobulins," American Biotech. Lab. 7, No. 2 16-19 (1989)]. It was demonstrated that rabbit anti-human IgG retained higher biological activity after binding to a hydrazide-containing solid support using AvidGel.TM. Ax.
Nonetheless, prior art methods exploiting the binding of immunoglobulins via their oligosaccharide moieties to hydrazide gels have heretofore required a relatively time-consuming and complicated procedure in which oxidation of the oligosaccharide moieties (typically, using sodium periodate) is invariably followed immediately by a separate process step for removal of unreacted periodate and any aldehydes released from the carbohydrate side chains. This separation procedure (typically, effected by column chromatography or dialysis) not only substantially increases the amount of time necessary to complete the binding procedure and greatly dilutes the sample, but may also reduce significantly the amount of valuable antibody recovered for binding. As the presence of any sodium periodate was considered to adversely effect the binding procedure, the conventional wisdom has been not to collect and pool fractions other than the protein peak, notwithstanding the potential loss and dilution of valuable antibodies in those peaks [see. e.g., Bio-Rad, "Affi-Gel.RTM. Hz Immunoaffinity Kit Instruction Manual, Catalog Number 153-6060, p. 5 (1987)].
In addition, it is highly desirable to effect the binding process as quickly as possible to reduce the amount of time during which immunoglobulins in an impure fraction may be subjected to protease and oxidative attack. The known methods for separation of sodium periodate from oxidized IgG using desalting gel column chromatography or dialysis are time-consuming and cause substantial dilution and/or loss of the sample. As a consequence, even after the recommended column chromatography it is generally necessary to perform sample concentration steps, which of course increase the processing time and the risk of denaturing the proteins and further loss of the sample.
It would be advantageous to provide a method for binding oxidized glycocompounds and glycoconjugates, and in particular glycoproteins such as valuable immunoglobulins, which could be carried out as rapidly as possible and in the least number of separate steps.
It is therefore an object of the present invention to provide a method for the binding of oxidized glycocompounds and glycoconjugates, and in particular glycoproteins, to hydrazide gels while obviating the problems inherent in the prior art methods.